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1 From the Department of Pathology, College of Medicine, University of Saskatchewan, and Cancer Research Unit, Health Research Division, Saskatchewan Cancer Agency, Saskatoon, Saskatchewan, Canada; and the 2 Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
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Abstract |
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METHODS. Whole bovine eyes were either homogenized for purification or regionally dissected to determine CaN localization and activity. Dissected tissues were homogenized and Western blot analysis performed, using polyclonal anti-CaN antibodies, and assayed using p-nitrophenyl phosphate (PNPP) as a substrate to determine the dephosphorylation activity of CaN. Fresh samples were then prepared for immunohistochemistry and probed with polyclonal anti-CaN antibodies.
RESULTS. CaN was found to be present in all eye tissues, although activity and
protein expression varied. The highest levels of CaN activity and
protein expression were found in the optic nerve, retina, and cornea.
Immunohistochemical methods displayed similar results with additional
staining of the optic nerve vasculature. Assays of purified CaN
demonstrated that bovine eye CaN had regulatory properties similar to
CaN isolated from other tissues. Probing eye tissues with CaN A
isoform-specific antibodies demonstrated that eye tissues displayed
variable distributions of the CaN A
and CaN Aß isoforms.
CONCLUSIONS. The presence of CaN in the bovine eye provides a physiological pathway by which the phosphorylated state of proteins and intracellular Ca2+ concentrations can be coordinated. The authors propose that CaN is involved in the immunologic privilege of the cornea, retinal signal transduction, and the toxic effects of immunosuppressants on the eye. Further in vivo studies of CaN function are necessary to understand the contributions of CaN to ocular physiology.
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Introduction |
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CaN is a heterodimer consisting of 19- and 57–59-kDa subunits referred to as CaN B and CaN A, respectively.13 14 CaN B contains four EF-hand Ca2+-binding sites15 and has affinity with CaN A. CaN A has four functional domains, including a CaN B binding domain, a calmodulin (CaM)-binding domain, an autoinhibitory domain, and the catalytic site.15 CaN is activated by CaM, a ubiquitously expressed protein involved in many Ca2+-dependent processes. The CaN A subunit has low endogenous phosphatase activity and requires Ca2+, CaM, and CaN B for full activity.15 The activity of CaN can be further stimulated by divalent metal cations such as Ni2+ and Mn2+ in vitro.16
The most clinically important feature of CaN is its selective inhibition by the immunosuppressants cyclosporine and FK506 (tacrolimus).17 Inhibition of CaN by immunosuppressants was first observed in T cells, and it was later discovered that many side effects of immunosuppressants on body organs can be attributed to inhibition of CaN in those tissues.18 The central protein regulated by CaN in immunosuppression is the nuclear factor of activated transcription (NF-AT). NF-AT is a cytosolic protein that is inactive in its phosphorylated state. The activation of T cells by receptor stimulation causes influx of Ca2+ into the cell. CaN is then fully activated, dephosphorylating NF-AT, thus allowing it to enter the nucleus and promote gene transcription.19 20 The dephosphorylated intranuclear NF-AT then promotes the expression of cytokines necessary for a rapid and effective T-cell response.20 It is the inhibition of CaN in this process that mediates the pharmacologic response seen in immunosuppression. This suppression of immune function has found clinical use in organ transplantation and in the treatment of certain inflammatory diseases.21 22 23
Originally discovered and purified as an inhibitor of CaM-dependent phosphodiesterase in bovine brain,24 25 CaN has since been characterized in numerous other tissues including kidney, liver, muscle, and T lymphocytes.16 18 20 Previous examination of CaN in the eye demonstrated its presence in chick retina26 and more recently in the developing and mature mammalian retina.27 To date, however, a comprehensive study of CaN throughout all tissues of the visual axis has not been undertaken, thus forming the rationale for the present study. We were able to identify and purify CaN from whole bovine eye extracts and also described the distribution of CaN by Western blot analysis and immunohistochemistry. By examining the localization, regulatory properties, and activity of CaN in all portions of the eye, we hoped to gain insight into the physiological roles CaN may play in maintaining mammalian ocular function or its contribution to disease states of the eye.
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Methods |
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Preparation of Crude Homogenates
Selected bovine eye tissues (cornea, lens, iris, ciliary body,
vitreous body, retina, choroid, sclera, and optic nerve) were dissected
and homogenized, using a homogenizing probe for 5 minutes in 2 mL
ice-cold 20 mM Tris-HCl buffer (pH 7.5), per gram of tissue. Crude
homogenates for purification were prepared from whole bovine eyes using
a meat grinder followed by homogenization in a blender for 1 minute in
the previously mentioned buffer containing 2 mM EDTA, 100 mg/L
phenylmethylsulfonyl fluoride, 100 mg soybean trypsin inhibitor, and
200 mg/L benzamidine. These protease inhibitors were included in the
buffers up to the CaM-Sepharose-4B affinity chromatography step. The
homogenates were centrifuged at 10,000g for 25 minutes, and
the supernatant was filtered through glass wool.
2-Mercaptoethanol and EGTA were added to the supernatants of samples to final concentrations of 10 and 0.1 mM, respectively.
Phosphatase Assay
CaN phosphatase activity was assayed using
p-nitrophenyl phosphate (PNPP; Sigma Chemical Co.) as a
substrate.28
Twenty-five micrograms of crude tissue
homogenate and buffer were added to the assay mixture to a total volume
of 1 mL. One unit of phosphatase activity was defined as the amount of
dephosphorylation resulting in an optical density of 0.1 at 30°C
after 30 minutes’ incubation. All assays were performed in duplicate
in three separate samples.
Production of Polyclonal Antibody against CaN
Antibodies were raised in New Zealand White rabbits given
multiple subcutaneous injections of 50 µg bovine brain CaN emulsified
with complete Freund’s adjuvant (CFA) at multiple sites. The first
blood samples were obtained on day 36, and two boosters were given with
the same amount of antigen in CFA, one on day 37 and the other on day
56. Subsequent blood samples were obtained on days 55 and 60. The sera
were separated from the blood samples and stored at -20°C until use.
The antibody titer was determined by ELISA, as described
earlier.29
The antibody was purified through a protein
A-Sepharose-4B column, pre-equilibrated with 100 mM Tris-HCl (pH 8.0),
followed by elution of bound IgG with 100 mM glycine (pH 2.5).
Immediately after elution, the pH was adjusted to 8.0 with 1 M Tris,
and the immunoglobulin was dialyzed against phosphate-buffered saline
(PBS) overnight. The antibodies were demonstrated to be specific for
CaN (data not shown). The dialyzed sample was stored at -70°C in
small aliquots containing 0.9 mg of protein per milliliter.
Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis and
Western Blot Analysis
SDS-PAGE (10%) was performed according to the method of
Laemmli.30
CaN A isoform-specific antibodies (A
and Aß) were obtained from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA). Western blot analyses were performed essentially as
described by Towbin.31
Samples were subjected to 10%
SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad Laboratories
Ltd., Mississauga, Ontario, Canada), and blocked with 5% milk powder
in PBS with Tween 20 (PBST) for 1 hour at room temperature. Polyclonal
and isoform-specific antibodies were diluted (1:1000) in blocking
buffer and incubated with the membrane overnight at 4°C. Membranes
were then washed once with PBST for 30 minutes and washed three times
for 5 minutes each to remove unbound antibody. Horseradish
peroxidase–conjugated secondary antibodies (Bio-Rad Laboratories Ltd.)
were diluted (1:2000) and incubated with membranes for 1 hour at room
temperature. Membranes were then washed once with PBST for 1 hour and
washed twice for 5 minutes each to remove unbound secondary antibody.
Immunoreactive bands were visualized on imaging film (Eastman Kodak
Co., Rochester, NY) using chemiluminescence reagents (NEN Life Sciences
Products, Inc., Boston, MA). Quantitative analysis of Western blot
analysis was performed using imaging software obtained from the
National Institutes of Health
(http://www.rsb.info.nih.gov/nih-image/download.html).
Immunohistology
Immunohistology was performed as described
previously.32
Bovine eyes, fixed in 10% formaldehyde and
dehydrated in ascending solutions of ethanol and xylene, were embedded
in paraffin. Five sections of 7-µm thickness were prepared from
tissue blocks and placed on slides coated with silane. The slides were
kept at 55°C for 45 minutes in an oven to improve adherence of
sections.
The sections were deparaffinized and rehydrated in xylene and descending concentrations of ethanol. Endogenous peroxidase was neutralized by a 30-minute incubation in 0.5% hydrogen peroxide in methanol. The antigens were unmasked by treating the sections with 2 mg pepsin per milliliter 0.01 N HCl for 45 minutes. This incubation time was determined with a series of trials and was found to yield maximum staining.
The sections were then blocked with 1% bovine serum albumin in PBS for 30 minutes, followed by incubation with anti-CaN polyclonal antibody (1:100) for 90 minutes and with HRP-conjugated secondary antibodies (1:100–1:400; Dako Corp., Carpinteria, CA) for 45 minutes. The reactions were color developed with a peroxidase substrate kit (SK4600; Vector Laboratories, Burlingame, CA). Control experiments included omission of either the primary antibody or both primary and secondary antibodies to determine, respectively, nonspecific binding of the secondary antibody and inhibition of endogenous tissue peroxidase. Some of the sections were stained with von Willebrand factor antibody (vWF; 1:200; Dako Corp.) to delineate blood vessels and to obtain another control. Some slides were counterstained for 2 to 4 minutes with methyl green. The slides were examined and images captured on an image analysis system (Northern Eclipse, Empix Imaging, Mississauga, Ontario).
Other Methods
Protein concentrations were determined by the method of Bradford
using bovine serum albumin as a standard.33
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Results |
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Activation of CaN by Divalent Metal Cations and Inhibition by CaM
Antagonists
Purified CaN was assayed with Ni2+ and CaM
to determine maximum in vitro activity. In the presence of
Ni2+ alone, CaN activity was 38%, compared with
the activity observed in the presence of Ni2+ and
CaM. CaN also displayed activation by other divalent cations such as
Mn2+ and Ca2+. The
antipsychotic drug fluphenazine has been shown to be a reversible
inhibitor of CaM, and its addition to reaction assays reduced CaN
activity to 57%, compared with similar reactions without fluphenazine.
In the absence of Ca2+, CaM, or metal cations,
the endogenous activity of CaN was found to be 5% of maximally
activated levels.
Determination of the CaN A Isoform
It has been shown that CaM-regulated enzymes exist in multiple
isoforms with unique functions and characteristics,35
36
37
38
and CaN A also has been demonstrated to exist in
isoforms.12
Because the eye is a heterogeneous organ
containing many different tissues, immunoblot analysis of CaN was
performed in different regions of the eye. The expression of the two
major isoforms of CaN A, CaN A and CaN Aß, were examined by
probing eye tissues with isoform-specific polyclonal antibodies. CaN
A and CaN Aß antibodies reacted with all eye samples examined with
the exception of samples from lens and cornea (Fig. 4)
. Lens tissue displayed no detectable reaction for either of the CaN A
isoforms, an observation also apparent in Figure 2
. Cornea however
displayed distinctive isoform expression. Strong reaction was observed
for the CaN A isoform in corneal samples (Fig. 4 , top), whereas
there was negligible expression of the CaN Aß isoform (Fig. 4
,
bottom).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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and CaN Aß, although both isoforms are not present in all eye
tissues. Purified bovine eye CaN displayed similar regulatory
properties as observed in CaN from other tissues.4
CaN in
bovine eye displayed stimulation by Ca2+, CaM,
and divalent cations.
Western blot analysis indicated that both subunits of CaN are expressed
in eye, and antibodies specific for two isoforms of CaN A cross-reacted
with various eye tissue samples. The implications for the expression of
CaN A isoforms stems from the distribution of these isoforms in other
tissues. The CaN Aß isoform is predominantly expressed in cells of
lymphoid lineage and is believed to be the isoform responsible for
mediation of the immune response.18
The CaN A isoform
is more widely distributed, and its inhibition is believed to cause
many of the detrimental side effects of
immunosuppression.18
The presence of both isoforms of CaN
A in eye suggests CaN may be involved in immunoregulation and other
presently unknown functions (Fig. 4)
. However the expression of only
the CaN A isoform in cornea is unique (Fig. 4 , top). The amount of
CaN determined by quantitative Western blot analysis (Fig. 2)
in the
eye further demonstrates that CaN is present in relatively low amounts,
compared with levels in other tissues.16
The recent study by Nakazawa et al.27
demonstrated CaN
distribution in developing rat and mature rat, bovine, and human
retina, and further demonstrated that distribution of CaN was limited
to the cell bodies of ganglion cells and cells of the inner nuclear
layer. Our study further confirms this distribution; however, when
probing with isoform and subunit-nonspecific antibody we found the
distribution of CaN to be more widespread within retinal tissue cell
types (Fig. 3G)
. Our observations agree with the study by Nakazawa et
al., in that we were able to demonstrate expression of both the CaN
A and CaN Aß isoforms in the retina (Fig. 4) .
The cornea is a unique tissue in many respects, the most noteworthy of which is its immune privilege,39 which allows for the high success rate of unmatched corneal transplantation.40 A recently discovered factor involved in corneal immune privilege is the constitutive expression of Fas ligand (Fas L) by corneal epithelial and endothelial cells.41 FasL, a transmembrane protein that is a member of the tumor necrosis factor family,42 is limited to activated T cells and to certain cells in the testes and cornea.40 The receptor for FasL (Fas+) conversely is more widely expressed, being found on the surface of many cells, including T-cells. The interaction between FasL and its receptor results in the forcing of the Fas+ cell to undergo apoptosis.43 The importance of FasL in the survival of corneal grafts is emphasized by experiments in mice with neither FasL nor Fas+, which demonstrate an increased rate of rejection of corneas taken from FasL-deficient mice and from hosts that do not have the Fas+ receptor.40
Recent studies have shown CaN activity to be a component that is necessary in the functional expression of FasL.6 We have shown in this study that corneal epithelial tissue expresses large quantities of CaN (Figs. 2A 3B) . It was also noted that CaN in cornea had an almost undetectable expression of the CaN B subunit (Fig. 2C) . The possibility of corneal CaN's being a unique isoform, perhaps with higher endogenous activity, is thus speculated and further investigation into this is warranted. The further possibility of the involvement of CaN in both ischemic11 and apoptotic8 9 processes in cornea, both of considerable significance, may also be of importance in future studies.
The response of retinal photoreceptor cells to light is a second potential pathway for regulation by CaN. During the photoreceptor cycle a series of reactions result in a net decrease of cGMP concentrations in photoreceptors, causing an inactivation of cGMP-gated cation channels that allows the photoreceptor to extrude ions and the return to a resting potential.44 One of the ions regulated in this process is Ca2+. Intracellular Ca2+ concentrations in photoreceptors are also mediated by release from intracellular stores by the interaction of IP345 46 47 with its receptor. CaN has been shown to regulate IP3 receptors in other tissues by dephosphorylation5 and leads us to hypothesize that Ca2+ concentrations in photoreceptors could also be regulated by CaN. These pathways involving CaN could suppress the amplitude of Ca2+ oscillations in photoreceptors facilitating a more rapid transition from the light- to the dark-adapted photoreceptor. Such a possible pathway could account for the relatively high amounts of CaN demonstrated in the retina (Fig. 3G) .
The immunosuppressants cyclosporine and FK506, both known selective inhibitors of CaN, are very effective in suppressing immune reactions but also have many systemic side effects, including ocular complications.48 49 50 Cyclosporine, which is neurotoxic,51 can cause cortical blindness and severe retinal disease.52 53 54 55 56 57 We were able to demonstrate expression of CaN in the vasculature of bovine eye (Fig. 3D) , thus supporting a possible ischemic mechanism of immunosuppressant toxicity. CaN is also involved in neuronal signal transmission and in the production and maintenance of myelin sheaths.2 58 The relatively low levels of CaN in eye may make it particularly susceptible to the side effects of the CaN inhibitors cyclosporine and FK506.
Our studies demonstrate the first characterization and localization of CaN in the entire eye. Expression of CaN in eye, as described in the present study, provides a framework for further studies on immunosuppressants and their ocular effects. Currently, research is being conducted into the development of synthetic corneas for use in corneal transplantation.59 Ensuring expression of CaN and FasL in these tissues will be an important factor in determining long-term survival of these grafts. Culturing conditions with sufficient Ca2+ concentrations to maintain CaN activity should add to the survival of these corneas. Furthermore, the potential future use of topical immunosuppressants in the treatment of eye disease and the role CaN plays in certain ocular pathologic states should be examined. Studies into the physiological function of CaN in vivo should be undertaken to examine these facets of ocular function.
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Acknowledgements |
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Footnotes |
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Submitted for publication March 30, 2001; revised July 26, 2001; accepted September 5, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Rajendra K. Sharma, Department of Pathology, College of Medicine, University of Saskatchewan, 20 Campus Drive, Saskatoon, SK, Canada S7N 4H4; rsharma{at}scf.sk.ca.
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